Use of fragments of oxytocin for the preparation of a pharmaceutical composition in order to create eustasis

ABSTRACT

The present invention relates to the use di-, tri- and tetrapeptide fragments of oxytocin or mesotocin for the preparation of a pharmaceutical composition in order to create eustasis. The invention also relates to di-, tri- and tetrapeptide fragments of oxytocin or mesotocin for medical use, as well as a pharmaceutical composition comprising at least one di-, tri- or tetrapeptide fragment of oxytocin or mesotocin in order to create eustasis.

The present invention relates to the use di-, tri- and tetrapeptidefragments of oxytocin or mesotocin for the preparation of apharmaceutical composition in order to create eustasis. The inventionalso relates to di-, tri- and tetrapeptide fragments of oxytocin ormesotocin for medical use, as well as a pharmaceutical compositioncomprising at least one di-, tri- or tetrapeptide fragment of oxytocinor mesotocin in order to create eustasis.

BACKGROUND OF THE INVENTION

Oxytocin was one of the first peptide hormones to be isolated andsequenced. It is a nonapeptide with two cysteine residues that form adisulfide bridge between positions 1 and 6 and corresponds to theformula

A similar nonapetide is mesotocin having the formula

For a long time the only effects attributed to oxytocin were itsstimulating effects on milk ejection and uterine contractions, but inthe past decades it has been shown that oxytocin exerts a wide spectrumof effects within the central nervous system, CNS. It has been suggestedthat oxytocin participates in the control of memory and learningprocesses and of various types of behaviour such as feeding, locomotion,as well as maternal and sexual behavior. Oxytocin is also suggested toparticipate in the control of cardiovascular functions,thermoregulation, and pain threshold and fluid balance. There is alsoevidence that oxytocin is involved in the control of variousimmunological processes. It has recently been demonstrated that oxytocininjections cause a lowering of blood pressure and increased weightgain—long lasting effects after repetitive administration. As a centralstimulating substance oxytocin plays an important role in theinteraction between mother and progeny in mammals. The products may alsobe used prophylactic in young human beings e.g. already in new bornbabies or young children to prevent the development of diseases later onin life which diseases are dependent on stress conditions during thefetal life. Such conditions may be heart/vessel diseases such as stroke,heart infarct, hypertension, and diabetes.

In the human body oxytocin is produced in the paraventricular nucleus,PVN, and the supraoptic nucleus, SON, of the hypothalamus. It differs byonly two amino acids from vasopressin, which is also produced in thesenuclei. The magnocellular oxytocinergic neurones of the SON and PVN sendoxons to the posterior pituitary from which oxytocin is released intothe circulation. Parvocellular neurones that originate in the PVNproject into multiple areas within CNS. The oxytocin-producing cells areinnervated by cholinergic, catecholaminergic as well as peptidergicneurones. The presence of oxytocin in different tissues outside thebrain, such as the uterus, ovaries, testis, thymus, adrenal medulla andpancreas has been demonstrated and oxytocin is suggested to exert localeffects in these organs.

A parallel secretion of oxytocin into the brain regions and into thecirculation occurs in response to some stimuli such as suckling, butother stimuli can cause separate activation of oxytocinergic neurones,terminating in the brain or the pituitary.

Mesotocin, which has been isolated from frogs, has similar effects asoxytocin.

The oxytocin and mesotocin molecule may be digested into smallerfragments. Of special interest are fragments containing 2-4 peptidesobtained by cleavage amino acids from the amino terminal and/or thecarboxyl terminal. As used in the context of the present invention, theterm “di-, tri- and tetrapeptide fragments of oxytocin or mesotocin”correspond to such fragments. Such fragments may either be amidated ornot at the C-terminal. It has now been shown that di-, tri- andtetrapeptide fragments of oxytocin or mesotocin may be used in order tocreate eustasis.

By the expression “eustasis” we understand a psychophysiological state,i e a combination of a psychological and physiological state. Thepsychological state is characterised by calm and positive socialinteractions such as trust and breast-feeding. The physiological stateis characterised by muscle relaxation, lowered cardiovascular activityand enhanced gastrointestinal activity. Besides, pulse rate and bloodpressure are kept at a low, healthy and balanced level, and the vagallycontrolled gastrointestinal tract is activated, promoting digestion andstorage of nutrients.

Eustasis should not be confused with euphoria, which is more an intensefeeling of joy and reward. Furthermore, the creation of eustasis shouldnot be confused with the treatment of depression or any other diseasestates. For example, the treatment of depression refers to a conversionfrom a disease state to a healthy state, whereas the creation ofeustasis refers to a conversion from one healthy state to anotherhealthy state.

In the Examples, some compounds according to the invention areadministered to rats. A decrease in locomotor activity, blood pressureand hormone levels was noticed, suggested that the compounds have aeustasis creating effect.

U.S. Pat. No. 5,767,083 (D1) discloses the use of Pro-Leu-Gly-NH₂ (SEQID NO: 8)and Pro-Ile-Gly-NH₂ (SEQ ID NO: 9). The first-mentionedoligopeptide is called melanocyte stimulating inhibitory factor,abbreviated MIF, and is used against depression. Furthermore, D1describes the combination of such oligopeptides known antidepressantssuch as amitriptyline, fluoxetine, and sertraline.

EP 146 113 (D2) discloses that Pro-Leu-Gly-NH₂ (SEQ ID NO: 9) may beused against disease states caused by elevated melanotropine levels,such as high blood pressure. D2 also discloses the dipeptide Leu-Gly-NH₂(SEQ ID NO: 17).

Regulatory Peptides From Molecular Biology to Function, Advances inBiochemical Psychopharmacology, Vol. 33, Ed. E. Costa and M. Trabucchi,Raven Press, New York (1982) (D3) discloses the effect of Leu-Gly-NH₂(SEQ ID NO: 17), acetic acid salt in the treatment of neuropsychiatricillness.

No documents disclose tetrapeptide fragments of oxytocin and mesotocin.As mentioned above, some di- and tripeptide fragments of oxytocin andmesotocin are previously known in the art. However, it is not previouslyknown to use di- and tripeptide fragments of oxytocin and mesotocin inorder to create eustasis. Besides, no pharmaceutical compositionscomprising the di- and tripeptides and oxytocin or mesotocin are knownin the art.

SUMMARY OF THE INVENTION

The present invention relates to the use of di, tri- and tetrapeptidefragments of oxytocin or mesotocin for the preparation of apharmaceutical composition in order to create eustasis. The inventionalso relates to di-, tri- and tetrapeptide fragments of oxytocin ormesotocin, preferably for medical use. It also relates to as apharmaceutical composition comprising at least one di-, tri- ortetrapeptide fragment of oxytocin or mesotocin, and a pharmaceuticallyacceptable carrier and, optionally oxytocin or mesotocin. Such apharmaceutical composition may be used in order to create eustasis.

DETAILED DESCRIPTION OF THE INVENTION

One object of the present invention is the use of a compound of theformula (I):

X₁-X₂-X₃-X₄ (SEQ ID NO: 3)

-   wherein X₁ is selected from Cys and nothing,-   X₂ is selected from Pro and nothing;-   X₃ is selected from Leu and Ile;-   X₄ is selected from Gly and nothing;-   provided that if X₁ is Cys, then X₂ is Pro;-   provided that if X₄ is nothing, then X₁ is Cys and X₂ is Pro;-   as well as amidated forms thereof;    for the preparation of a pharmaceutical composition, optionally    comprising oxytocin or mesotocin, in order to create eustasis.

By “nothing” is meant that the letters respectively may have no meaningor may represent a bond and that there may be a direct bond between theitems (letter, atom or group) situated to the right and to the left,respectively, of the letter designating “nothing”. For example, informula (I), when X₁ designates nothing, the resulting moleculecorresponds to X₂-X₃-Gly. When X₄ designates nothing, the resultingmolecule corresponds to X₁-X₂-X₃.

By “amidated” form is meant that the carboxylic acid group of thecarboxyl terminal amino acid residue of the resulting peptide isamidated.

It is preferred that the compound of formula (I) is selected from:

Cys-Pro-Leu-Gly (SEQ ID NO: 4) Cys-Pro-Leu-Gly-NH₂, (SEQ ID NO: 5)Cys-Pro-Ile-Gly, (SEQ ID NO: 6) Cys-Pro-Ile-Gly-NH₂, (SEQ ID NO: 7)Pro-Leu-Gly, (SEQ ID NO: 8) Pro-Leu-Gly-NH₂, (SEQ ID NO: 9) Pro-Ile-Gly,(SEQ ID NO: 10) Pro-Ile-Gly-NH₂, (SEQ ID NO: 11) Cys-Pro-Leu, (SEQ IDNO: 12) Cys-Pro-Leu-NH₂ (SEQ ID NO: 13) Cys-Pro-Ile, (SEQ ID NO: 14)Cys-Pro-Ile-NH₂ (SEQ ID NO: 15) Leu-Gly, (SEQ ID NO: 16) Leu-Gly-NH₂,(SEQ ID NO: 17) Ile-Gly (SEQ ID NO: 18) and Ile-Gly-NH₂. (SEQ ID NO: 19)

It is also preferred that the compound is administered in an amount of 1μg to 1 mg/kg body weight of the patient.

Another object of the present invention is a compound of the formula(I):

X₁-X₂-X₃-X₄ (SEQ ID NO: 3)

-   wherein X₁ is selected from Cys and nothing,-   X₂ is selected from Pro and nothing;-   X₃ is selected from Leu and Ile;-   X₄ is selected from Gly and nothing;-   provided that if X₁ is Cys, then X₂ is Pro;-   provided that if X₄ is nothing, then X₁ is Cys and X₂ is Pro;-   as well as amidated forms thereof;-   provided that if X₁ is nothing, X₂ is Pro, then X₄ is Gly in    non-amidated form; and-   provided that if X₁ and X₂ are nothing, and X₃ is Leu, then X₄ is    Gly in non-amidated form.

By “non-amidated” form is meant that the carboxylic acid group of thecarboxyl terminal amino acid residue of the resulting peptide is notamidated.

It is preferred that the compound of formula (I) is selected from:

Cys-Pro-Leu-Gly (SEQ ID NO: 4) Cys-Pro-Leu-Gly-NH₂, (SEQ ID NO: 5)Cys-Pro-Ile-Gly, (SEQ ID NO: 6) Cys-Pro-Ile-Gly-NH₂, (SEQ ID NO: 7)Pro-Leu-Gly, (SEQ ID NO: 8) Pro-Ile-Gly, (SEQ ID NO: 10) Cys-Pro-Leu,(SEQ ID NO: 12) Cys-Pro-Leu-NH₂ (SEQ ID NO: 13) Cys-Pro-Ile, (SEQ ID NO:14) Cys-Pro-Ile-NH₂ (SEQ ID NO: 15) Leu-Gly, (SEQ ID NO: 16) Ile-Gly(SEQ ID NO: 18) and Ile-Gly-NH₂. (SEQ ID NO: 19)

Another object of the present invention is a compound as defined abovefor medical use.

Another object of the present invention is a pharmaceutical compositioncomprising at least one compound as defined above, and apharmaceutically acceptable carrier and, optionally, oxytocin ormesotocin, in order to create eustasis.

It is preferred that the effective concentration of the compound in thepharmaceutical composition is 4-70% by weight, preferably 0.1-50% byweight.

The invention also relates to the peptides mentioned above in both D-and L-form. Especially the invention relates to the L-form. By inversionof the peptide sequence thereof, the D-form could be converted to theL-form. The effect of the D- and L-forms are the same. These and thepeptides above can be produced by methods known to a person skilled inthe art e.g. according to Merrifield, P. B., “Solid Phase Synthesis”,Angew. Chemie, 1985, No. 97, p. 801.

The pharmaceutical compositions are prepared in a manner known to aperson skilled in the pharmaceutical art. The carrier or the excipientcould be a solid, semi-solid or liquid material that could serve as avehicle or medium for the active ingredient. Suitable carriers orexcipients are known in the art. The pharmaceutical composition could beadapted to oral, parenteral, intravaginal, or topical use and could beadministered to the patient as tablets, capsules, suppositories,solutions, suspensions or the like.

The pharmaceutical compositions could be administered orally, e.g. withan inert diluent or with an edible carrier. They could be enclosed ingelatine capsules or be compressed to tablets. For oral therapeuticadministration the compounds according to the invention could beincorporated with excipients and used as tablets, lozenges, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain at least 4% by weight of the compoundsaccording to the invention, the active ingredient, but could be variedaccording to the special form and could, suitably, be 4-70% by weight ofthe unit. The amount of the active ingredient that is contained incompositions is so high that a unit dosage form suitable foradministration is obtained.

The tablets, pills, capsules, lozenges and the like could also containat least one of the following adjuvants: binders such asmicrocrystalline cellulose, gum tragacanth or gelatine, excipients suchas starch or lactose, disintegrating agents such as alginic acid,Primogel, corn starch, and the like, lubricants such as magnesiumstearate or Sterotex, glidants such as colloidal silica dioxide, andsweetening agents such as saccharose or saccharin could be added orflavourings such as peppermint, methyl salicylate or orange flavouring.When the unit dosage form is a capsule it could contain in addition tothe type above a liquid carrier such as polyethylene glycol or a fattyoil. Other unit dosage forms could contain other different materialsthat modify the physical form of the unit dosage form, e.g. as coatings.Accordingly, tablets or pills could be coated with sugar, shellac orother enteric coating agents. A syrup could in addition to the activeingredient contain saccharose as a sweetening agent and somepreservatives, dyes and flavouring agents. Materials that are used forpreparation of these different compositions should be pharmaceuticallypure and non-toxic in the amounts used.

For parental administration the compounds according to the inventioncould be incorporated in a solution or suspension. Parenteraladministration refers to the administration not through the alimentarycanal but rather by injection through some other route, as subcutaneous,intramuscular, intraorbital, intracapsular, intraspinal, intrasternal,intravenous, intranasal, intrapulmonary, through the urinary tract,through eye drops, rectal or intravaginal (e.g. as a suppository, avagitorium, a cream or an ointment), through the lactiferous tract incattle, into an organ such as bone marrow, etc. Bone marrow may also betreated in vitro. These preparations could contain at least 0.1% byweight of an active compound according to the invention but could bevaried to be approximately 0.1-50% thereof by weight. The amount of theactive ingredient that is contained in such compositions is so high thata suitable dosage is obtained.

The solutions or suspensions could also comprise at least one of thefollowing adjuvants: sterile diluents such as water for injection,saline, fixed oils, polyethylene glycols, glycerol, propylene glycol orother synthetic solvents, antibacterial agents such as benzyl alcohol ormethyl paraben, antioxidants such as ascorbic acid or sodium bisulfite,chelating agents such as ethylene diamine tetraacetic acid, buffers suchas acetates, citrates or phosphates, and agents for adjustment of thetonicity such as sodium chloride or dextrose. The parenteral preparationcould be enclosed in ampoules, disposable syringes or multiple dosagevessels made of glass or plastic.

For topical administration the compounds according to the inventioncould be incorporated in a solution, suspension, or ointment. Thesepreparations could contain at least 0.1% by weight of an active compoundaccording to the invention but could be varied to be approximately0.1-50% thereof by weight. The amount of the active ingredient that iscontained in such compositions is so high that a suitable dosage isobtained. The administration could be facilitated by applying touch,pressure, massage, heat, warms, or infrared light on the skin, whichleads to enhanced skin permeability. Hirvonen, J., Kalia, Y N, and Guy,R H. Transdermal delivery of peptides by iontophoresis, Nat Biotechnol1996 December; 14(13): 1710-1713 describes how to enhance the transportof a drug via the skin using the driving force of an applied electricfield. Preferably, iontophoresis is effected at a slightly basic pH.

Other administration forms are inhalation through the lungs, buccaladministration via the mouth, enteral administration via the smallintestine, and local administration with a release, preferably a slowrelease, of the active substance e g in the form of a ring. All theseadministration forms could be effected by means known by a personskilled in the art.

All publications mentioned herein are hereby incorporated by reference.By the expression “comprising” we understand including but not limitedto. Thus, other non-mentioned substances, additives or carriers may bepresent.

The invention will be illuminated by the following Examples, which areonly intended to illuminate and not restrict the invention in any way.

EXAMPLES

Materials and Methods

Animals. Eight week-old male Sprague-Dawley rats (280-300 g) wereobtained from B&K Universal AB, Sollentuna, Sweden.

Experimental procedure. SEQ ID NO: 5 was dissolved in physiologicalsaline (1 mg/ml) and administered subcutaneously to six rats at a doseof 1 mg/kg for five consecutive days. Six rats received physiologicalsaline only as a control. Two days after the last treatment, the ratswere exposed to blood pressure determination with the cuff technique.The locomotor behaviour was studied three days after the end of thetreatment. The next day the animals were decapitated and blood wascollected.

Statistics. The results are presented as means ±SD. Statistical analysiswas performed by means of 2-way ANOVA followed by Bonferroni's test forpost hoc comparison. A Students t-test was performed. p-values of 0.05or less were regarded as statistically significant.

Example 1 Locomotor Activity Observations

The spontaneous motor activity was observed in a square open-field arena(680×680×450 mm), equipped with two rows of photocells (8×8), sensitiveto infrared light. Two identical frames of photocells were placed at twolevels, 40 and 125 mm above the floor, respectively. The open-field wasenclosed in a ventilated, sound-attenuating box with a Perspex top.Measurements were made in the dark and performed between 0.900-16.00 h.

The number of photocell beam interruptions were collected on anIBM-compatible PC computer allowing the registration of locomotoractivity (all interruptions of photobeams at the lower level) andrearing (all interruptions of photobeams at the upper level). The datawere subject to a square root transformation. For further details on theapparatus and the computer software used, including a schematic drawingof the equipment, see Ericson, E., Samuelsson, J., and Ahlenius, S.(1991). Photocell measurements of rat motor activity: A contribution tosensitivity and variation in behavioral observations. J Pharmacol Meth25, 111-122. Furthermore, the peripheral activity was measured. Byperipheral activity is meant that the animals are located in the cornersof the arena. When the animals are frightened, they have a highertendency to be located in the corners ie have a high peripheralactivity. On the contrary, when the animals are more calm, they have ahigher tendency to be located in the middle of the arena ie have a lowerperipheral activity.

Results

The locomotor activity was significantly decreased in rats treated withSEQ ID NO: 5, as well as the amount of rearing. Likewise, the amount ofperipheral activity decreased. Together, these data show that thecompounds according to the invention induce a calming effect.

Example 2 Plasma Hormone Determinations

Trunk blood was collected in ice-chilled tubes, containing heparin (10IU/ml) (Pharmacia-Upjohn, Stockholm, Sweden) and aprotinin (500 IU/ml)(Trasylol, Bayer, Germany). The blood samples were centrifuged andplasma was removed and frozen (−20° C.).

Oxytocin

Oxytocin was measured with a specific radioimmunoassay developed in thislaboratory (Stock, S., and Uvnäs-Moberg, K. (1988). Increased plasmalevels of oxytocin in response to stimulation of the sciatic and vagalnerves and in response to touch and pinch in anaesthetized rats. ActaPhysiol Scand 132, 29-34.). Plasma samples were purified withreversed-phase chromatography using C18 Waters SEP-PAK cartridges. Theantiserum anti-oxytocin (rabbit) for RIA, KA-19 (Euro Diagnostica,Malmö, Sweden) and the tracer [¹²⁵I]-Tyr²-oxytocin (Du Pont Nen ResearchProducts, Boston, Mass.) were used. The cross-reactivity of the antibodywas less than 0.01% with vasopressin, somatostatin LH-RH and ACTH. Thelimit of detection was 2 pmol/l. The intra- and interassay coefficientsof variation were 11 and 13%, respectively.

Gastrin

Gastrin was immunoassayed as described by Smedh, U., and Uvnäs-Moberg,K. (1994) Intracerebroventricularly administered corticotropin-releasingfactor releases somatostatin through a colinergic, vagal pathway infreely fed rats. Acta Physiol Scand 151, 241-247. The gastrin standard(Pensinsula Laboratories), the antiserum No 260 4 (gift fom J F Rehfeld,Rigshospitalet, Copenhagen Denmark) and the tracer [¹²⁵I]-gastrin 8 EuroDiagnostic, Malmö Sweden were used. The reactivity of the antibody hadthe same potency for gastrin-17 and gastrin-34. The limit of detectionof the assay was 6 pmol/l. The intra- and interassay coefficients ofvariation were 10 and 13%, respectively.

Cholecystokinin (CCK)

CCK was immunoassayed as described by Smedh and Uvnäs Moberg, supra.Plasma samples were purified with reversed-phase chromatography usingC18 Waters SEP PAK cartridges. The CCK standard (Peninsula Laboratories)the antiserum OAL 656 (Otsuka Assay Laboratories, Japan) and the tracer[¹²⁵I]-CCK (Du Pont NEN Research Products Boston, Mass.) were used. Theantiserum OAL 656 was raised against N-terminal amino acid residue ofsulphated CCK 8 and reacted with CCK 8, CCK 39 but not with gastrin andits related peptides. The limit of detection of the assay was 3-6 pmol/lThe intra- and interassay coefficients of variation were 10 and 12%respectively.

Results

The animals treated for five consecutive days with SEQ ID NO: 5 had thehormone levels as shown in Table 1.

TABLE 1 Hormone levels of rats treated with SEQ ID NO: 5 compared tocontrol rats. Hormone levels in hormone treated Hormone levels inHormone rats (N = 6) control rats (N = 6) p-value Oxytocin 29.7 ± 7.2 53.6 ± 14.3 0.004 Gastrin  85 ± 19.3  130 ± 52.6 0.09 CCK 8.4 ± 3.5 14.6± 7.6  0.06

These data indicate that the compounds according to the inventionsignificantly lower the oxytocin levels, whereby the cholinergic vagalmechanisms are influenced. This suggest a stimulating influence ondigestion and anabolic metabolism.

Example 3 Measurement of Blood Pressure and Heart Rate

Blood pressure and heart rate were measured on conscious animals byplacing a cuff (Kent RTBP-002, Somedic Sales, Farsta, Sweden) on thebase of the tail. The cuff was connected to a Grass 7P8 sphygmanometerand a Grass 7P8DC amplifier with a printer. The rats were habituated tothe entire test procedure for 2-3 weeks before the procedure started.

Results

The blood pressure was significantly decreased in rats treated with SEQID NO: 5. The systolic and diastolic blood pressures are given in Table2.

TABLE 2 Hormone levels of rats treated with SEQ ID NO: 5 compared tocontrol rats. Value in hormone Value in control Blood pressure treatedrats (N = 6) rats (N = 6) p-value Systolic 116 ± 9.1  132.1 ± 9.2  0.01Diastolic 87.3 ± 18.9 104.9 ± 14.8 0.01

1. A peptide consisting of the amino acids selected from the groupconsisting of: (SEQ ID NO: 14) and (SEQ ID NO: 15).
 2. A method forrelaxing muscles, lowering cardiovascular activity, pulse rate, bloodpressure, and to enhance gastrointestinal activity in a patient in needof treatment thereof, comprising administering an effective amount ofpeptide according to claim 1 to said patient.
 3. A pharmaceuticalcomposition comprising: at least one peptide according to claim 1; and apharmaceutically acceptable carrier.
 4. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, at least one ofoxytocin and mesotocin, and a peptide according to claim
 1. 5. Thepharmaceutical composition according to claim 3, wherein the peptide is4-70% by weight of the composition.
 6. The pharmaceutical compositionaccording to claim 3, wherein the peptide is 0.1-50% by weight of thecomposition.
 7. The pharmaceutical composition according to claim 4,wherein the peptide is 4-70% by weight of the composition.
 8. Thepharmaceutical composition according to claim 4, wherein the peptide is0.1-50% by weight of the composition.